The major aim of this project is to resolve mechanisms controlling cell fate decisions in developing T cells. Signals through the TCR and other surface receptors determine whether thymocytes will survive, mature, or die. Precursor T cells undergo a testing process in the thymus to ensure that cells expressing useless or self-reactive TCRs do not mature. These selection processes (known as positive and negative selection) require TCR engagement with peptide-MHC ligands expressed by thymic stromal cells. Thymocytes also receive signals as they mature that direct them to specific lineages. Signals through the gamma-delta TCR or the pre-TCR determine whether early T cell precursors will specify the gamma-delta or alpha-beta T cell fate. Immature thymocytes that choose the alpha-beta pathway of T cell development are signaled through the TCR to mature as CD4 helper T cells or as CD8 cytotoxic T cells. Our studies and those of others indicate that it is the strength and/or duration of TCR signaling during the process of positive selection that direct this lineage choice. In addition, T cell fate may be determined by the integration of TCR signals with other developmental cues, such as those regulated by the Notch receptor. Although less well understood, such environmental signals are involved also in the developmental fate of B, NK, NK T, regulatory T, and H2-M3-restricted T cell lineages in the thymus. [unreadable] Over the last year, we have continued our efforts to understand the role of Notch signaling in thymocyte development and, recently, in the differentiation of peripheral T cell lineages; and to a lesser degree, in the development of non-T cell lineages in lymphoid tissues. Since there are four Notch family members, our first priority has been to establish experimental models in which all Notch signaling is abrogated in developing T cells. We have accomplished this aim with conditional, double null mutations of Presenilin1/Presenilin2, which are proteins that are indispensable for mediating all Notch activation. In this project, we generated combinations of standard and conditional gene-targeted knock-out mice; some with TCR and other transgenes, bred onto several selecting MHC backgrounds (often RAG-deficient). Using different Cre transgenes to mediate gene deletion in specific tissues, we have observed all the phenotypic defects previously associated with Notch mutations in lymphoid development; but in addition, we find abnormalities in dendritic cells and in late stages of thymocyte development not previously reported. This year we completed a study showing that Presenilin-deficiency mediated by CD4-Cre reduces the number of mature CD4 T cells. We find that the generation of CD4+ thymocytes from CD4+CD8+ (DP) precursors is inefficient in mice with a diverse TCR repertoire, and severely compromised in mice expressing a single MHC class II-restricted TCR. Defective CD4 T cell development is associated with impaired TCR signaling at the DP stage as indicated by a reduced calcium response to TCR stimulation in vitro and decreased CD5 upregulation in response to selecting MHC in vivo. The fact that CD4 T cell generation can be somewhat compensated in Presenilin-deficient mice by higher affinity or density of selecting MHC ligand demonstrated that defective T cell maturation was a consequence of poor positive selection. Moreover, the number and phenotype of DP thymocytes was corrected by expression of a constitutively active form of Notch, indicating that the phenotypes observed in Presenilin-deficient thymocytes were due to a loss of Notch signaling. Given these results, we would like to understand how the TCR and Notch signaling pathways are functionally linked in thymocytes. We have compared gene expression in mutant and normal thymocytes by microarray analyses and are examining other components of TCR signal transduction for additional defects. Although Notch appears to directly regulate binary fate decisions in many systems, these findings suggest instead a model in which Notch signaling influences positive selection and the development of mature T cells by modifying TCR signal transduction. Notably, this is the only in vivo model in existence which prevents all Notch signaling in late thymocyte maturation. Although CSL is thought to be major transcriptional mediator of Notch signaling, we found that the conditional CSL mutant models (developed by the Honjo lab) do not promote sufficiently early and/or efficient gene deletion, so cannot be used to assess the role of Notch in late T cell maturation. [unreadable] Because Amyloid Precursor Protein (APP) family members can also serve as substrates for Presenilins, it was important to study their expression in the thymus. Recently, we have completed a project which is the first comprehensive analysis of expression and function of APP family proteins in lymphoid tissues. These studies show APP and Amyloid Precursor-like Protein 2 (APLP 2) are expressed by stromal cells of thymus and lymph nodes, but not by lymphocytes. Although signals provided by thymic stromal cells are critical for normal T cell differentiation, lymphocyte development proceeds unperturbed in mice deficient for either or both of these proteins. Thus, these studies indicate that inhibitors that specifically block cleavage of APP family members (used to treat Alzheimers Disease) are unlikely to have immunosuppressive side effects.[unreadable] In other efforts to understand how TCR signals promote cell fate decisions in the thymus, we have focused on the pre-TCR that promotes survival, alpha-beta T cell commitment, proliferation, differentiation, and allelic exclusion at an early stage of thymocyte development. In one study, we have focused on the invariant pre-Talpha chain that with a newly rearranged TCRbeta forms the pre-TCR heterodimer. Pre-Talpha is a transmembrane protein consisting of an immunoglobulin extracellular domain and an intracellular 30 amino acid proline rich tail. Although the proline rich tail is unique both for its length and intrinsic signaling motifs, the contribution of the cytoplasmic tail to pre-TCR signaling and function has not been defined. To study the signaling potential of the pre-Talpha tail under normal physiological regulation, we have used knock-in technology to induce a truncation of the pre-Talpha locus which generates a mutant pre-Talpha protein lacking the intracellular tail. In these mutant mice, alpha-beta versus gamma-delta T lineage commitment occurs normally. Although surface expression and internalization/ degradation of the pre-TCR containing the tailless pre-TCRalpha were normal; fewer thymocytes, a partial block in differentiation, and a perturbation in cell cycle kinetics and proliferation were observed. Moreover, when mutant progenitors were placed in direct competition with normal progenitors in hematopoietic chimeras, mutant precursors were significantly less efficient than normal precursors in reconstituting the irradiated thymus. Introducing a TCRbeta transgene for dimerization with the tailless pre-Talpha revealed that this mutant form of pre-TCR inefficiently suppresses endogenous TCRbeta rearrangements, compared with a complete loss of the full length pre-Talpha in which allelic exclusion is completely abrogated. Collectively, these results support a quantitative rather than a qualitative role for the cytoplasmic tail in functions associated with pre-TCR signaling in early thymocytes.